Lipoproteins are globular, micelle-like particles that consist of a non-polar core of acylglycerols and cholesteryl esters surrounded by an amphiphilic coating of protein, phospholipid, and cholesterol. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons, which transport dietary lipids from intestine to tissues; very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL), all of which transport triacylglycerols and cholesterol from the liver to tissues; and high density lipoproteins (HDL), which transport endogenous cholesterol from tissues to the liver.
Lipoprotein particles undergo continuous metabolic processing and have variable properties and compositions. Lipoprotein densities increase without decreasing particle diameter because the density of their outer coatings is less than that of the inner core. The protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human lipoproteins.
Apolipoprotein C-III is a constituent of HDL and triglyceride-rich lipoproteins and has a role in hypertriglyceridemia, a risk factor for coronary artery disease. Apolipoprotein C-III slows the clearance of triglyceride-rich lipoproteins by inhibiting lipolysis, both through inhibition of lipoprotein lipase and by interfering with lipoprotein binding to the cell-surface glycosaminoglycan matrix (see, Shachter, Curr. Opin. Lipidol., 12:297-304 (2001)).
The gene encoding human apolipoprotein C-III (also called APOC3 and apoC-III) was cloned in 1984 (see, Levy-Wilson et al., DNA, 3:359-364 (1984); Protter et al., DNA, 3:449-456 (1984); Sharpe et al., Nucleic Acids Res., 12:3917-3932 (1984)). The coding sequence is interrupted by three introns (see, Protter et al., supra). The human APOC3 gene is located approximately 2.6 kilobases to the 3′ direction of the apolipoprotein A-1 gene and these two genes are convergently transcribed (see, Karathanasis, Proc. Natl. Acad. Sci. U.S.A., 82:6374-6378 (1985)). Also cloned was a variant of the human APOC3 gene resulting in a Thr74 to Ala74 mutation from a patient with unusually high levels of serum apoC-III protein. As the Thr74 is O-glycosylated, the Ala74 mutant therefore resulted in increased levels of serum apoC-III protein lacking the carbohydrate moiety (see, Maeda et al., J. Lipid Res., 28:1405-1409 (1987)).
Five polymorphisms have been identified in the promoter region of the APOC3 gene: C(-641) to A; G(-630) to A; T(-625) to deletion; C(-482) to T; and T(-455) to C. All of these polymorphisms are in linkage disequilibrium with the SstI polymorphism in the 3′ untranslated region. The SstI site distinguishes the S1 and S2 alleles and the S2 allele has been associated with elevated plasma triglyceride levels (see, Dammerman et al., Proc. Natl. Acad. Sci. U.S.A., 90:4562-4566 (1993)). The APOC3 promoter is downregulated by insulin and this polymorphic site abolishes insulin regulation. Thus, the potential overexpression of apoC-III resulting from the loss of insulin regulation may be a contributing factor to the development of hypertriglyceridemia associated with the S2 allele (see, Li et al., J. Clin. Invest., 96:2601-2605 (1995)). The T(-455) to C polymorphism has been associated with an increased risk of coronary artery disease (see, Olivieri et al., J. Lipid Res., 43:1450-1457 2002)).
In addition to insulin, other regulators of APOC3 gene expression have been identified. A response element for the nuclear orphan receptor rev-erb alpha has been located at positions −23/−18 in the APOC3 promoter region and rev-erb alpha decreases APOC3 promoter activity (see, Raspe et al., J. Lipid Res., 43:2172-2179 (2002)). The APOC3 promoter region −86 to −74 is recognized by two nuclear factors, CIIIb1 and CIIIB2 (see, Ogami et al., J. Biol. Chem., 266:9640-9646 (1991)). APOC3 expression is also upregulated by retinoids acting via the retinoid X receptor, and alterations in retinoid X receptor abundance affects APOC3 transcription (see, Vu-Dac et al., J. Clin. Invest., 102:625-632 (1998)). Specificity protein 1 (Sp1) and hepatocyte nuclear factor-4 (HNF-4) have been shown to work synergistically to transactivate the APOC3 promoter via the HNF-4 binding site (see, Kardassis et al., Biochemistry, 41:1217-1228 (2002)). HNF-4 also works in conjunction with SMAD3-SMAD4 to transactivate the APOC3 promoter (see, Kardassis et al., J. Biol. Chem., 275:41405-41414 (2000)).
Transgenic and knockout mice have further defined the role of apoC-III in lipolysis. Overexpression of APOC3 in transgenic mice leads to hypertriglyceridemia and impaired clearance of VLDL-triglycerides (see, de Silva et al., J. Biol. Chem., 269:2324-2335 (1994); Ito et al., Science, 249:790-793 (1990)). Knockout mice with a total absence of apoC-III protein exhibited significantly reduced plasma cholesterol and triglyceride levels compared with wild-type mice and were protected from postprandial hypertriglyceridemia (see, Maeda et al., J. Biol. Chem., 269:23610-23616 (1994)).
Recently, it was discovered that about 5% of the Lancaster Amish are heterozygous carriers of a null mutation in exon 3 of the APOC3 gene consisting of a C to T transition at nucleotide 55, resulting in an Arg19 to Ter (R19X) substitution (see, Pollin et al., Science, 322:1702-1705 (2008)). As the mutation occurs in the signal peptide of the protein, a complete lack of production of apoC-III from alleles carrying the mutation was predicted. Carriers of the R19X null mutation expressed half the amount of apoC-III present in noncarriers. Mutation carriers compared with noncarriers had lower fasting and postprandial serum triglycerides, higher levels of HDL cholesterol, and lower levels of LDL cholesterol. Subclinical atherosclerosis, as measured by coronary artery calcification, was less common in carriers than noncarriers, which suggested that lifelong deficiency of apoC-III protein has a cardioprotective effect.
In view of the foregoing, there is a need for therapeutic agents capable of effectively inhibiting APOC3 function and methods for their in vivo delivery to target tissues such as the liver. The present invention addresses these and other needs.